The Cullin-ROC Family of E3 Ubiquitin Ligases

描述（由申请人提供）：这是GM 067113的首次竞争性更新，该研究旨在了解E3泛素连接酶（CRL）cullin-RING家族的功能和调控的长期目标。回顾过去几年的历程，我们很高兴看到我们所做的贡献，更值得注意的是，同事们共同取得的进步。在最初发现ROC 1和ROC 2之后，我们已经朝着这个目标取得了一些发现。其中包括：（1）CAND 1，一种进化上保守的cullin相关和neddylation解离蛋白，控制底物向所有CRL的募集，（2）CUL 3与存在于超过200种哺乳动物蛋白中的BTB基序结合，并潜在地组装大量BTB-CUL 3-ROC 1连接酶，（3）DDB 1-CUL 4-ROC 1连接酶靶向复制许可因子CDT 1以响应DNA损伤而降解，（4）CUL 4与大量细胞蛋白相互作用，（5）p170（KIAA 080），一种进化上保守的WD 40蛋白，通过DDB 1与CUL 4A大量结合，并以p53依赖的方式在细胞增殖中起重要作用。在此基础上，我们提出了进一步研究CUL 3和CUL 4依赖性泛素连接酶的底物募集机制、细胞功能和调控的设想。本提案中概述的研究结合了联合收割机计算机建模、蛋白质组学、生物化学、细胞和遗传学方法，为帮助了解E3泛素连接酶的最大家族提供了极好的和独特的机会。三个具体目标是：（i）确定CRLS在有丝分裂和转录调节中的功能和底物，（ii）确定CRL 4连接酶的底物募集机制，和（iii）确定p170的功能和机制。

DESCRIPTION (provided by applicant): This is the first competitive renewal of GM067113, an investigation aimed at the long term goal of understanding the function and regulation of cullin-RING family of E3 ubiquitin ligases (CRLs). Looking back at the journey of the past several years, we are very pleased to see the contribution we made and more remarkably, the advancements achieved collectively by colleagues. Following the initial discovery of ROC1 and ROC2, we have made several findings toward this goal. These include: (1) that CAND1, an evolutionary conserved cullin-associated and neddylation dissociated protein, controls substrate recruitment to all CRLs, (2) that CUL3 binds to a BTB motif present in more than 200 mammalian proteins and potentially assembles a large number of BTB-CUL3-ROC1 ligases, (3) that DDB1-CUL4-ROC1 ligase target the replication licensing factor CDT1 for degradation in response to DNA damage, and (4) that CUL4 interacts with large number of cellular proteins, and (5) that pl70(KIAA080), an evolutionarily-conserved WD40 protein, binds abundantly with CUL4A via DDB1 and plays an essential role in cellular proliferation in a p53-dependent manner. With these series of findings as the basis, we propose a further research to elucidate the substrate- recruitment mechanism, the cellular function and regulation of CUL3- and CUL4-dependent ubiquitin ligases. The studies outlined in this proposal combine computer modeling, proteomic, biochemical, cellular and genetic approaches, offering an excellent and unique opportunity to help in the understanding of the largest family of E3 ubiquitin ligases. Three specific aims are: (i) to determine the functions and substrates of CRLS in mitosis and in transcriptional regulation, (ii) to determine the substrate recruiting mechanism of CRL4 ligases, and (iii) to determine the function and mechanism of p170.